Pathobiology of an NS1-Truncated H3N2 Swine Influenza Virus Strain in Pigs

ABSTRACT Virus strains in the live attenuated influenza vaccine (LAIV) for swine in the United States that was on the market until 2020 encode a truncated nonstructural protein 1 of 126 amino acids (NS1del126). Their attenuation is believed to be due to an impaired ability to counteract the type I interferon (IFN)-mediated antiviral host response. However, this mechanism has been documented only in vitro for H3N2 strain A/swine/Texas/4199-2/98 NS1del126 (lvTX98), and several cases of clinical respiratory disease in the field were associated with the LAIV strains. We therefore further examined the pathobiology, including type I IFN induction, of lvTX98 in pigs and compared it with IFN induction in pig kidney-15 (PK-15) cells. lvTX98 induced up to 3-fold-higher type I IFN titers than wild-type TX98 (wtTX98) after inoculation of PK-15 cells at a high multiplicity of infection, while virus replication kinetics were similar. Mean nasal lvTX98 excretion by intranasally inoculated pigs was on average 50 times lower than that for wtTX98 but still reached titers of up to 4.3 log10 50% tissue culture infective doses/mL. After intratracheal inoculation, mean lvTX98 titers in the lower respiratory tract were significantly reduced at 18 to 48 h postinoculation (hpi) but similar to wtTX98 titers at 72 hpi. lvTX98 caused milder clinical signs than wtTX98 but induced comparable levels of microscopic and macroscopic lung lesions, peak neutrophil infiltration, and peak type I IFN. Thus, lvTX98 was partly attenuated in pigs, but this could not be associated with higher type I IFN levels. IMPORTANCE Swine influenza A viruses (swIAVs) with a truncated NS1del126 protein were strongly attenuated in previous laboratory-based safety studies and therefore approved for use as LAIVs for swine in the United States. In the field, however, the LAIV strains were detected in diagnostic samples and could regain a wild-type NS1 via reassortment with endemic swIAVs. This suggests a significant degree of LAIV replication and urges further investigation of the level and mechanism of attenuation of these LAIV strains in vivo. Here, we show that H3N2 LAIV strain lvTX98 is only partly attenuated in pigs and is excreted at significant titers after intranasal vaccination. Attenuation and restricted replication of lvTX98 in vivo seemed to be associated with the loss of NS1 functions other than type I IFN antagonism. Our findings can help to explain the occurrence of clinical respiratory disease and reassortment events associated with NS1del126-based LAIV strains in the field.

IMPORTANCE Swine influenza A viruses (swIAVs) with a truncated NS1del126 protein were strongly attenuated in previous laboratory-based safety studies and therefore approved for use as LAIVs for swine in the United States. In the field, however, the LAIV strains were detected in diagnostic samples and could regain a wild-type NS1 via reassortment with endemic swIAVs. This suggests a significant degree of LAIV replication and urges further investigation of the level and mechanism of attenuation of these LAIV strains in vivo. Here, we show that H3N2 LAIV strain lvTX98 is only partly attenuated in pigs and is excreted at significant titers after intranasal vaccination. Attenuation and restricted replication of lvTX98 in vivo seemed to be associated with the loss of NS1 functions other than type I IFN antagonism. Our findings can help to explain the occurrence of clinical respiratory disease and reassortment events associated with NS1del126-based LAIV strains in the field. KEYWORDS attenuation, H3N2, influenza A, NS1, pathobiology, swine, type I interferon I nfluenza A virus (IAV) is a major cause of respiratory disease in pigs and results in significant economic losses in the swine industry. The disease is difficult to control due to the complex swine IAV (swIAV) epidemiology: within the H1 and the H3 subtypes, multiple antigenically different lineages and clades circulate simultaneously (1,2). swIAVs of each clade continue to evolve due to accumulation of point mutations, which occur mainly in the immunodominant hemagglutinin (HA) surface protein (antigenic drift) (3). In addition, swIAVs can evolve by the exchange of RNA gene segments MOI of 2 (Fig. 1A). Mean lvTX98 virus titers were on average 1.0 log 10 TCID 50 /mL lower than those of wtTX98 after inoculation at low MOI (P , 0.05 at 10 to 48 hpi) but similar to wtTX98 titers after inoculation at high MOI. No bioactive IFN-a/b was detected in PK-15 cell supernatants after inoculation with wtTX98 at a low MOI of 0.001, whereas lvTX98 induced IFN-a/b titers of up to 9.5 log 2 U/mL in 3/5 replicates of the experiment at this MOI (Fig. 1B). After inoculation at a high MOI of 2, wtTX98 induced detectable bioactive IFN-a/b titers of up to 7.2 log 2 U/mL in 2/5 replicates of the experiment, while lvTX98 consistently induced IFN-a/b with peak titers that were 3-fold higher than those induced by wtTX98 (P , 0.05 at 18 to 30 hpi). Thus, in PK-15 cells, lvTX98 replicates to lower titers than wtTX98 after inoculation at a low MOI and is a better IFN-a/b inducer.
Reduced nasal excretion of lvTX98 compared to wtTX98 after i.n. inoculation of pigs. In previous studies, i.n. inoculation of pigs with lvTX98 resulted in nasal excretion at mean titers below 1 log 10 TCID 50 /mL (10), and pigs showed minimal clinical signs (7,17). However, after commercialization of the Ingelvac Provenza vaccine containing lvTX98, the number of diagnostic field cases associated with the LAIV strains increased (https:// influenza.cvm.iastate.edu/). This suggests substantial in vivo lvTX98 replication and shedding. To evaluate nasal lvTX98 excretion, pigs were inoculated intranasally with 6.3 log 10 TCID 50 of lvTX98 or wtTX98 and clinical signs as well as virus titers in nasal swabs were evaluated at 0 to 7 days postinoculation (dpi). The inoculation dose was selected based on previous vaccination studies with lvTX98 (9)(10)(11)(12)(13). None of the pigs showed clinical signs during the experiment. lvTX98 virus could be detected in nasal swabs until 6 dpi, while wtTX98 virus was detected until 7 dpi. Nasal lvTX98 excretion was on average 1.7 log 10 TCID 50 /mL lower than wtTX98 excretion at 1 to 6 dpi, and the differences were statistically significant (P , 0.05, Fig. 2). However, lvTX98 was still excreted at substantial titers: the mean lvTX98 virus titer reached 4.3 log 10 TCID 50 /mL at 3 dpi. This contrasts with previous studies indicating minimal lvTX98 shedding (10).
lvTX98 causes milder clinical signs than wtTX98 after i.t. inoculation of pigs. The considerable lvTX98 excretion after i.n. inoculation of pigs raises questions about the safety of lvTX98. We therefore compared the pathogenicity of this virus in pigs with that of wtTX98. To reproduce the typical clinical signs observed in the field under FIG 1 wtTX98 and lvTX98 replication and IFN-a/b-inducing capacity in PK-15 cells. PK-15 cell monolayers were infected with a MOI of 0.001 or a MOI of 2 of wtTX98 (blue circles) or lvTX98 (orange squares), and supernatant samples were evaluated for virus titers in a CPE assay on MDCK cells (A) and for bioactive IFN-a/b titers in a CPE reduction assay with VSV on MDBK cells (B) at different time points postinoculation. Means with standard deviations of log 10 -transformed virus titers, log 2 -transformed IFN-a/b titers, and areas under the curve of 5 replicates are shown. Black dotted lines represent the detection limit. Results were compared between groups using 2-sided Mann-Whitney U tests. *, P , 0.05. experimental conditions, pigs were inoculated with a high dose of 7.5 log 10 TCID 50 of wtTX98 or lvTX98 via the intratracheal (i.t.) route (5). Pigs were scored for clinical signs from 96 h before inoculation (296 hpi) until 72 hpi as shown in Table 1. Rectal temperatures and breathing rates were the most important parameters determining the overall clinical score.
Before inoculation, none of the pigs showed significant clinical signs (Tables 2 and  3). One pig had slight fever at 224 hpi, and some pigs showed occasional tachypnea or sneezing. Coughing, conjunctivitis, and depression were recorded only once. Inoculation with wtTX98 or lvTX98 each led to increased overall clinical scores at 18 to 72 hpi, but peak mean scores were 3 times higher with wtTX98 than with lvTX98.
At 18 hpi with wtTX98, 9/12 pigs had fever with rectal temperatures up to 40.5°C and 10/12 pigs had tachypnea with breathing rates up to 56 respirations (resp.)/min. Clinical signs peaked at 24 hpi: rectal temperatures and breathing rates increased to up to 41.7°C and 64 resp./min, respectively; 8/9 pigs had fever; and all pigs had tachypnea. In addition, several pigs showed labored abdominal breathing, depression, coughing, and sneezing. Fever had disappeared by 48 hpi, but most of the former clinical signs as well as dyspnea and tachypnea were still detected. At 72 hpi, only breathing rates were still elevated.
After inoculation with lvTX98, clinical signs already peaked at 18 hpi. Overall clinical scores, rectal temperatures, and breathing rates were significantly lower than at 24 hpi with wtTX98 (P = 0.002, 0.003, and 0.03, respectively). Only 3/12 pigs had fever with temperatures up to 41.4°C, and 8/12 pigs had tachypnea with breathing rates up to 64 resp./min. Some pigs also showed labored abdominal breathing and sneezing. At 24 hpi, these signs as well as coughing were recorded, 1 pig still had fever, and 8/9 pigs had tachypnea. At 48 to 72 hpi, only tachypnea and depression were detected.
Peak clinical signs after i.t. inoculation were thus less abundant and less severe with lvTX98 than with wtTX98. Although lvTX98 could still cause clinical signs, it was attenuated compared to the wild-type virus.
lvTX98 can induce similar pathological changes in the lower respiratory tract as wtTX98. At different time points after i.t. inoculation, 3 pigs per group were sacrificed to examine trachea and lung lesions as well as neutrophil infiltration in the lungs caused by lvTX98 and wtTX98. For the latter, cells in bronchoalveolar lavage (BAL) fluid were stained and counted (Table 4).
Macroscopic lesions at the lung surface were detected in both the wtTX98 and the lvTX98 group (Fig. 3A), mainly on the diaphragmatic lung lobes. Gross lung lesions involved up to 17% of the lungs with wtTX98 and up to 10% with lvTX98. Differences between groups were not significant, except at 18 hpi, when on average 2% more macroscopic lung lesions were detected in lvTX98-inoculated pigs (P = 0.08).
Microscopic trachea lesions were absent in both groups, except for 1 pig with focal epithelial attenuation at 72 hpi with wtTX98. In the lungs, microscopic lesions were FIG 2 wtTX98 and lvTX98 excretion by intranasally inoculated pigs. Conventional influenza-naive pigs were intranasally inoculated with 6.3 log 10 TCID 50 of wtTX98 (blue circles, n = 6) or lvTX98 (orange squares, n = 16) in 3 mL of PBS, and nasal swabs were taken daily to evaluate virus titers in a CPE assay on MDCK cells. Log 10 -transformed virus titers of individual pigs are shown by dots; lines indicate mean values of each group. Virus titers were compared between groups using 2-sided Mann-Whitney U tests. The black dotted line represents the detection limit. *, P , 0.05; **, P , 0.001. present in 10/12 pigs inoculated with wtTX98 and in only 3/12 pigs inoculated with lvTX98 at 18 to 72 hpi ( Fig. 3B and Table 5). wtTX98 caused epithelial damage in up to 75% of the airways, peribronchiolar lymphocytic cuffing in up to 50% of the airways, and minimal to large aggregates of neutrophils in bronchiolar lumens. Similarly, lvTX98 caused epithelial damage in up to 25% of the airways, no to $75% peribronchiolar lymphocytic cuffing, and small to large aggregates of neutrophils in bronchiolar lumens of the 3 affected pigs. Only at 18 hpi, composite scores were significantly lower with lvTX98 than with wtTX98 (P = 0.06).
Both viruses caused infiltration of neutrophils in the lungs (Fig. 3C). In the wtTX98 group, 2/3 pigs had $10% neutrophils in the lungs at 18 hpi. At 24 hpi, a peak in both macrophages and neutrophils was seen for all 3/3 pigs and BAL fluid cells consisted of 13 to 20% neutrophils. In the lvTX98 group, neutrophil infiltration peaked at 18 hpi, when 2/3 pigs had 14 to 25% neutrophils in BAL fluid. Peak percentages of neutrophils in BAL fluid, at 18 hpi with lvTX98 and at 24 hpi with wtTX98, were not significantly different between the two groups (P = 0.8).
In summary, lvTX98 was only partly attenuated in terms of lung pathology, as it caused pathological changes less frequently than but at similar severity as wtTX98.
lvTX98 replicates in both the upper and the lower respiratory tract of pigs. Virus titration was performed on various parts of the respiratory tract collected at different time points after i.t. inoculation. Mean lvTX98 titers were generally lower than mean wtTX98 titers, except at 24 hpi in the nose and at 72 hpi in cell-free BAL fluid (Fig. 4). Variation between pigs was, however, large, and hash marks in Fig. 4 indicate the single time points where all pigs in the lvTX98 group had lower titers than all pigs in the wtTX98 group (P # 0.1). Significant differences in the lower respiratory tract were found at 18 to 48 hpi, when mean lvTX98 titers were on average 3.6 log 10 lower than mean wtTX98 titers (Fig. 4B). In the upper respiratory tract, significant differences were found later, at 48 to 72 hpi, and mean virus titers were on average only 2.2 log 10 lower for lvTX98 than for wtTX98 (Fig. 4A). Importantly, lvTX98 could replicate to substantial titers of up to 5.8 log 10 TCID 50 /g in most respiratory tissues. By 72 hpi, lvTX98 titers were comparable to wtTX98 titers in the lower respiratory tract and the nasopharynx. Thus, lvTX98 replication occurred in all parts of the respiratory tract and was not consistently restricted. lvTX98 attenuation cannot be related to higher cytokine levels in vivo. lvTX98 has been shown to induce higher levels of IFN-a/b than wtTX98 in the continuous PK-15 cell line, but the kinetics of IFN-a/b secretion in the respiratory tract of pigs have never been examined. Therefore, BAL fluids of pigs inoculated intratracheally with either wtTX98 or lvTX98 were evaluated for levels of IFN-a/b as well as other proinflammatory cytokines, interleukin 6 (IL-6) and tumor necrosis factor a (TNF-a), which were previously shown to be important in swIAV pathogenesis (19).
Both wtTX98 and lvTX98 induced IFN-a, IFN-b, and IL-6 in pig lungs. Unlike in PK-    Pathobiology of H3N2 Influenza Virus lvTX98 in Pigs Journal of Virology 15 cells, bioactive IFN-a/b levels in BAL fluids of pigs tended to be lower after inoculation with lvTX98 than after inoculation with wtTX98 (Fig. 5A). The highest IFN-a/b titer in the lvTX98 group was 4 times lower than that in the wtTX98 group. Separate measurements of IFN-a and IFN-b were obtained using enzyme-linked immunosorbent assays (ELISAs). IFN-a ELISA titers reflected the results of the IFN-a/b bioassay and were generally lower in the lvTX98 group than in the wtTX98 group (Fig. 5B). Unlike for IFN-a, the highest IFN-b ELISA titer was detected in a pig of the lvTX98 group (Fig. 5C). Similarly, the highest bioactive IL-6 titer was detected in a pig of the lvTX98 group and was 4 times higher than the highest IL-6 titer in the wtTX98 group (Fig. 5D). However, pigs in the lvTX98 group did not have systematically higher or lower IFN-a, IFN-b, and IL-6 titers than those in the wtTX98 group at the peak of cytokine induction, that is, at 18 hpi for lvTX98 and at 24 hpi for wtTX98. Peak IFN-a, IFN-b, and IL-6 titers were thus not significantly different between the two groups (P = 0.2). lvTX98 and wtTX98 did not consistently induce bioactive TNF-a as measured using  In summary, lvTX98 was not able to induce significantly higher peak levels of IFN-a, IFN-b, IL-6, or TNF-a than wtTX98 in the lungs of i.t.-inoculated pigs. lvTX98 attenuation could therefore not be associated with differential cytokine induction in vivo.
lvTX98 did not obtain additional mutations in vivo that were associated with reversion to virulence. Before the start of the experiments, MinION whole-genome sequencing of the lvTX98 and wtTX98 virus stocks used for inoculation was performed to confirm their genetic constellation. The sequences of the wtTX98 stock were identical to the reference sequences in GenBank, except for synonymous nucleotide mutation G259A in the polymerase acid protein (PA) gene. The NS1 sequence of the lvTX98 stock had a 78-nucleotide (nt) deletion and an insertion of the sequence TAG ATCT TGA T TAA T TAA as previously described (15). The rest of the lvTX98 sequences were identical to the reference sequences in GenBank, except for synonymous nucleotide mutation T1016C in the HA gene and nt mutation A87G resulting in amino acid mutation D27G in the PA gene. Based on analyses in the FluSurver database, the latter was not previously linked to increased virus replication or virulence. Because our in vivo results showed less attenuation and higher replication of lvTX98 than previously described, we hypothesized that lvTX98 might have undergone mutations in vivo which increased its replication potential and/or virulence compared to the lvTX98 virus used for inoculation. Therefore, whole-genome sequencing was also performed on nasal swabs taken 3 days after i.n. inoculation and on cell-free BAL fluids of pigs sacrificed at 3 days after i.t. inoculation. Viral sequences in these samples were identical to those of the virus used for inoculation. Thus, lvTX98 did not undergo nucleotide or amino acid mutations that could explain its higher replication potential and virulence in this study compared to previous studies. a Intrapulmonary airway epithelium (IPAW) scores. 0.0, no significant lesions; 1.0, a few airways affected, with bronchiolar epithelial damage; 1.5, more than a few airways affected (up to 25%); 2.0, 50% of airways affected, often with interstitial pneumonia; 2.5, approximately 75% of airways affected, usually with significant interstitial pneumonia; 3.0, more than 75% of airways affected, usually with significant interstitial pneumonia. b Peribronchiolar lymphocytic cuffing (PBLC) scores. 0.0, no significant lesions; 1.0, a few airways with light PBLC; 1.5, more than a few airways with PBLC (up to 25%); 2.0, 50% of airways with PBLC; 2.5, approximately 75% of airways with PBLC; 3.0, more than 75% of airways with PBLC. c Scores of neutrophil exudation in bronchioles and alveoli (Neutro). 0, no to minimal presence of neutrophils; 1, small clusters of neutrophils present in occasional airways; 2, prominent small to large aggregates of neutrophils in bronchiolar lumens, with minimal aggregates in alveoli.

DISCUSSION
Our results show that the NS1-truncated H3N2 strain in the LAIV that was available for swine in the United States from 2017 until 2020, lvTX98, is attenuated in pigs: clinical signs were present but milder and lung pathological changes were equally severe as but less frequent than those with wtTX98. This contrasts with the findings of a previous pathogenesis study in which lvTX98 induced no or only minimal clinical signs and lung lesions (15). The latter study, however, used a 220-fold-lower inoculation dose than in our study, which could explain the differences. Although lvTX98 nasal shedding and replication in the swine respiratory tract were restricted compared to those with wtTX98, they were still substantial. Titers in BAL fluids were similar for the two viruses by 3 dpi. In contrast, previous studies showed only minimal lvTX98 shedding after i.n. vaccination (10,18) and significantly lower lvTX98 titers than wtTX98 titers in BAL fluids at 4 to 5 dpi upon i.t. inoculation (15). These differences might be due to the lower inoculation dose in the latter study, a less sensitive method for evaluating virus shedding in previous studies, and high levels of MDA against lvTX98 in one study (18). We verified that lvTX98 induces higher levels of bioactive type I IFN than wtTX98 in PK-15 cells, although differences between IFN-a/b titers induced by the two swIAVs were smaller than previously reported (15). Our study is the first to show that lvTX98 induces similar levels of IFN-a/b as wtTX98 in swine lungs in vivo. Attenuation and restricted replication of lvTX98 in swine are therefore not associated with higher type I IFN induction.
The finding that lvTX98 induces higher levels of IFN-a/b in PK-15 cells in vitro but not in swine lungs in vivo is likely due to the unsuitability of PK-15 cells as a model for the in vivo situation. PK-15 cells most likely synthesize and secrete type I IFN after recognition of viral double-stranded RNA (dsRNA), a by-product of virus replication (20,21). Although the same IFN pathway may apply in infected airway epithelial cells, the main sources of IFN-a/b during IAV infections in vivo are macrophages and, more importantly, plasmacytoid dendritic cells (pDCs) (20)(21)(22). Porcine pDCs, also known as natural interferon-producing cells (NIPCs), are probably the most potent IFN-producing cells. They are present at very low numbers in the bloodstream but can migrate to the lungs upon IAV infection and flood the area with type I IFN (20,21,23). In contrast to other nucleated cells, NIPCs can be stimulated to rapidly produce large amounts of IFN-a/b by viral glycoprotein structures, independent of viral replication (21). Thus, the majority of IFN-a/b detected in the pig lung is likely produced by cell types and pathways that are not represented in PK-15 cell cultures in vitro. Another explanation might be that lvTX98 replication was less restricted in PK-15 cells than in swine. In PK-15 cells, lvTX98 replicated to similar titers as wtTX98 and induced higher levels of IFN-a/b, indicating that lvTX98 has a higher intrinsic IFN-inducing capacity than wtTX98. In swine lungs, lvTX98 replicated to much lower titers than wtTX98 at the time of cytokine induction. As IFN-a/b induction is generally viral dose dependent (24), the lower replication in combination with the higher intrinsic IFN-inducing capacity of lvTX98 may result in the induction of similar IFN-a/b levels as those for wtTX98 in vivo. Similarly, a highly virulent mouse-adapted H1N1 IAV with a complete NS1 deletion had a stronger IFN-inducing capacity than the IAV with a partial NS1 deletion in vitro, but restricted in vivo replication of the former resulted in comparable IFN induction levels by the two IAVs in mouse lungs (25).
Since lvTX98 induces similar IFN-a/b levels as wtTX98 in swine lungs, its attenuation and restricted replication in vivo are likely due to the loss of NS1 functions other than antagonism of the type I IFN response. Indeed, type I IFN in the lungs of IAVinfected pigs positively correlates with both virus titers and clinical signs (19), indicating a role in disease rather than attenuation. In addition, NS1 does not seem to be a potent inhibitor of type I IFN induction, as evidenced by the high IFN-a/b levels in the lungs of wtTX98-infected pigs. NS1del126 protein levels in PK-15 cells after lvTX98 infection were much lower than NS1 protein levels after wtTX98 infection (15) and the NS1del126 protein of mouse-adapted strains, which was sufficient to suppress IFN-b induction in vitro (26,27), was unstable in vivo due to the lack of the C-terminal domain (27). Therefore, the NS1del126 protein of lvTX98 is likely unstable and thus present at only minimal levels both in vitro and in vivo. Since IAVs lacking NS1 replicated about 100 times less well than the parental virus, even in Vero cells that are unable to produce IFN (24,28,29), NS1 functions other than interference with the type I IFN system of the host are important for viral replication and virulence. Attenuation and restricted replication of lvTX98 in pigs may be due to the elimination of NS1 effects such as inhibition of host protein synthesis as well as stimulation of viral RNA polymerase and translation of viral proteins (16).
Attenuation and immunogenicity of NS1del126 IAVs such as lvTX98 were previously observed in mice, ferrets, poultry, horses, macaques, and pigs, and they were therefore considered suitable LAIV candidates (8,(30)(31)(32). However, lvTX98 attenuation is obtained by alteration of only 1 gene segment, allowing the virus to regain a wild-type gene constellation via reassortment with IAVs that are endemic in the vaccinated swine herd. Since reassortment is facilitated by high virus replication, and based on the lvTX98 virus titers observed in this study, lvTX98 might not be sufficiently attenuated to use as an LAIV strain in the field. Indeed, after Ingelvac Provenza was licensed in the United States in 2017, the diagnostic cases submitted to the Iowa State University Veterinary Diagnostic Laboratory with an HA related to vaccine strains lvTX98 (H3N2) and lvMN99 (H1N1) increased to 3.946% and 2.857% in 2019, respectively, compared to ,0.1% in 2016. The number of cases again decreased to ,1% in 2020 to 2021, after withdrawal of the Ingelvac Provenza vaccine from the market (https://influenza.cvm.iastate.edu/) (33). Clinical signs and lung lesions consistent with IAV infection were reported for these cases, although a role for viral and/or bacterial coinfections could not be excluded (14). Reassortment between LAIV and endemic IAV strains was confirmed for a subset of these samples, and most reassortants obtained a wild-type NS1 with a selective advantage. One isolate had an additional deletion of 185 nucleotides in its NS1 (14) and might therefore have had an increased virulence (15). Collectively, these data indicate safety concerns about the NS1del126-based Ingelvac Provenza LAIV and support the withdrawal of the vaccine from the market in 2020.
In summary, our results show that lvTX98 pathogenicity is less restricted than indicated by previous reports. Unlike in vitro, higher type I IFN induction by lvTX98 than by wtTX98 was not observed in swine lungs. lvTX98 attenuation in vivo is more likely caused by low NS1del126 protein stability and the loss of NS1 functions unrelated to IFN. We found considerable lvTX98 replication in the swine respiratory tract and substantial nasal excretion of lvTX98 by vaccinated pigs, which can facilitate reassortment with endemic IAV strains and reintroduction of the historic vaccine strains in the swine population. These data for Ingelvac Provenza H3N2 LAIV strain lvTX98 question the safety of NS1del126-based LAIVs. Cells and viruses. PK-15 cells were grown in minimal essential medium (MEM with GlutaMAX; Gibco) supplemented with 10% fetal calf serum (FCS; Sigma) and antibiotics (100 IU/mL penicillin, 50 mg/mL streptomycin, 50 mg/mL gentamicin; Gibco). MDCK cells were cultured in MEM with 10% FCS, 1 mg/mL lactalbumin (BD Biosciences), and antibiotics. MDBK cells were grown in Dulbecco's modified Eagle's medium (DMEM with GlutaMAX; Gibco) supplemented with 10% FCS, 10 mM sodium pyruvate (Gibco), and antibiotics. B9 cells were maintained in Iscove's modified Dulbecco's medium (IMDM with GlutaMAX; Gibco) containing 5% FCS, 5 Â 10 25 M b-mercaptoethanol (Sigma), 20 pg/mL recombinant human interleukin-6 (IL-6; R&D Systems), 100 IU/mL penicillin, and 50 mg/mL streptomycin. PK-15 subclone 15 cells were cultured in MEM with 7% FCS, 100 IU/mL penicillin, 50 mg/mL streptomycin, and 1% nonessential amino acids (Gibco).

MATERIALS AND METHODS
Swine influenza A viruses A/swine/Texas/4199-2/1998 (wtTX98) and A/swine/Texas/4199-2/1998 NS1del126 (lvTX98) were generated via reverse genetics as previously described (15) and kindly provided by the National Animal Disease Center, Ames, IA, USA. lvTX98 has a 39 deletion in the NS1 gene and produces a truncated NS1 protein consisting of only the first 126 amino acids. wtTX98 and lvTX98 swIAV stocks were grown in MDCK cells or in the allantoic cavity of 10-day-old embryonated chicken eggs. Vesicular stomatitis virus (VSV) for IFN-a/b bioassays was grown in MDBK cells.
Infection of PK-15 cells. Three-to 4-day-old PK-15 monolayers in 6-well plates were washed once with phosphate-buffered saline (PBS) with Ca 21 and Mg 21 and then infected with wtTX98 or lvTX98 at an MOI of 0.001 or an MOI of 2 in MEM supplemented with 0.6% bovine serum albumin (Sigma), 1 to 2 mg/mL trypsin (Sigma), and antibiotics (infection medium). After 1 h of incubation (37°C, 5% CO 2 ), the unbound virus was washed away with PBS containing Ca 21 and Mg 21 , 3.3 mL of infection medium was added per well, and 300 mL supernatant from each well was sampled immediately for virus and IFN-a/b titration. This time point corresponded to 0 hpi. The cells were then further incubated at 37°C and 5% CO 2 , and samples for virus and IFN-a/b titration were taken at different time points postinoculation by collecting 300 mL supernatant per well and then adding 300 mL fresh infection medium. Cells inoculated with an MOI of 0.001 were sampled at 0, 6, 10, 18, 24, 30, and 48 hpi. Cells inoculated with an MOI of 2 were sampled at 0, 2, 4, 6, 8, 10, 18, 24, 30, and 48 hpi. Five replicates of the experiment were performed.
Infection of pigs. Conventional 4-week-old pigs (n = 52) were purchased from a commercial farm that was free of influenza A virus. Pigs of different experimental groups were housed in separate high-efficiency particulate air (HEPA)-filtered biosafety level 2 animal units. At arrival, pigs were treated with a single dose of ceftiofur antibiotic (Naxcel; Zoetis) to reduce bacterial contamination. Pigs were confirmed to be seronegative in hemagglutination inhibition assay (34)  To evaluate lvTX98 and wtTX98 excretion, 5-week-old pigs were intranasally inoculated with 6.3 log 10 TCID 50 of lvTX98 (n = 16) or wtTX98 (n = 6) in 3 mL Dulbecco's PBS (DPBS; Gibco); 1.5 mL was applied in each nostril using a canula. At 0 to 7 dpi, pigs were monitored for clinical signs and nasal swabs (1 swab per nostril) were taken for virus titration and sequencing. Pigs were then further used in another experiment. Because of the experimental design of the latter, different numbers of pigs were inoculated with lvTX98 versus wtTX98. Afterward, all pigs were humanely euthanized with a lethal dose of sodium pentobarbital (Kela NV).
To examine the pathogenesis of lvTX98 and wtTX98, 2 groups of 15 6-week-old pigs were used. In each group, 3 pigs were left noninoculated and were humanely euthanized with a lethal dose of sodium pentobarbital at 0 hpi. The 12 remaining pigs were inoculated intratracheally with 7.5 log 10 TCID 50 of lvTX98 or wtTX98 in 3 mL DPBS; inocula contained ,1.32 endotoxin units per mL in the Limulus assay (Lonza). At 18, 24, 48, and 72 hpi, 3 inoculated pigs per group were humanely euthanized with a lethal dose of sodium pentobarbital. From 4 days before inoculation until the end of the experiment, each pig was scored for clinical signs according to the scoring system shown in Table 1, and a composite score was calculated for each pig at each time point (296, 272, 248, 224, 0, 18, 24, 48, and 72 hpi). At each time point of euthanasia (0, 18, 24, 48, and 72 hpi), nasal swabs were collected (1 swab per nostril) for virus titration and sequencing. At necropsy, lungs were scored for macroscopic lesions and different parts of the respiratory tract were sampled for virus titration, histopathology, sequencing, and cytokine analysis. Virus titration was performed on samples from trachea, nasopharynx, nasal mucosa olfactory part, nasal mucosa respiratory part, left apical and cardiac lung lobe, and left diaphragmatic lung lobe. Histopathological analysis was performed on a sample from trachea and left diaphragmatic lung lobe. The right lung was used to collect BAL fluid for BAL cell and cytokine analysis, virus titration, and sequencing.
BAL and BAL cell analysis. The right lung was flushed with 80 mL of cold DPBS using a blunt 18gauge needle inserted through the trachea. BAL fluids (42 to 60 mL) were centrifuged for 10 min at 400 Â g and 4°C to separate the cells from cell-free BAL fluid. BAL cells were resuspended in DPBS, and their number and viability were determined using Türk's solution and trypan blue. Cytocentrifuge preparations (2 per pig) were made with 300 mL of a BAL cell suspension of 5 Â 10 5 cells/mL and stained with DiffQuik (Medion Diagnostics) to determine the number of neutrophils and macrophages. Forty milliliters of cell-free BAL fluid from each pig was ultracentrifuged for 90 min at 100,000 Â g to remove virus, after which it was concentrated 20 times using Centricon Plus-70 centrifugal filter units with a cutoff of 10 kDa (Millipore). This concentrated BAL fluid was used for cytokine analysis. The leftover cell-free BAL fluid was used for virus titration and sequencing.
Pathological examination of lungs. The percentage of gross pneumonia was determined by visual inspection of the lung surface, and tissue samples for histopathological examination of microscopic lesions were processed and scored as previously described (12).